CCM signaling complex (CSC) couples both classic and non-classic Progesterone receptor signaling

Background Breast cancer, the most diagnosed cancer, remains the second leading cause of cancer death in the United States, and excessive Progesterone (PRG) or Mifepristone (MIF) exposure may be at an increased risk for developing breast cancer. PRG exerts its cellular responses through signaling cascades involving classic, non-classic, or combined responses by binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs). Currently, the intricate balance and switch mechanisms between these two signaling cascades remain elusive. Three genes, CCM1-3, form the CCM signaling complex (CSC) which mediates multiple signaling cascades. Methods Utilizing molecular, cellular, Omics, and systems biology approaches, we analyzed the relationship among the CSC, PRG, and nPRs/mPRs during breast cancer tumorigenesis. Results We discovered that the CSC plays an essential role in coupling both classic and non-classic PRG signaling pathways by mediating crosstalk between them, forming the CmPn (CSC-mPRs-PRG-nPRs) signaling network. We found that mPR-specific PRG actions (PRG + MIF) play an essential role in this CmPn network during breast cancer tumorigenesis. Additionally, we have identified 4 categories of candidate biomarkers (9 intrinsic, 2 PRG-inducible, 1 PRG-repressive, 1 mPR-specific PRG-repressive, and 2 mPR-responsive) for Luminal-A breast cancers during tumorigenesis and have confirmed the prognostic application of RPL13 and RPL38 as intrinsic biomarkers using a dual validation method. Conclusions We have discovered that the CSC plays an essential role in the CmPn signaling network for Luminal-A breast cancers with identification of two intrinsic biomarkers. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-022-00926-z.

containing 10% FBS. Non-invading cells remaining on the upper surface of the membrane were removed using a cotton swab, and the invading cells on the bottom surface were stained with Diff-Quick stain kit (Fisher). Invading cells were visualized using 20X (T47D cells) or 40X (MCF7 cells) magnification on a Nikon microscope and photographed with a high-resolution digital camera. The invasion value was quantified from the mean number of five different fields per condition. Each experiment was repeated three times.

Immunohistochemistry (IHC) and immunofluorescence (IF)
Deparaffinization of paraffin-embedded tissue sections for IHC: Breast cancer tissue slides were purchased from various suppliers (BioChain and US Biomax) and were baked at 60°C for 2 hrs. Once cooled, the sections were washed 3X in xylene for 5 min each, followed by 3-min sequential washes in 100, 95, 90, 80 and 70% ethanol and then soaked in water before an antigen retrieval step.
Growth of T47D cells using chamber slides for IF applications: IF staining methods were performed as previously described [2]. Briefly, cells were grown to confluency and then treated with MIF+PRG (20 µM each) over time in Glass chamber Slides (Nunc-Lab-Tek II) and fixed using 4% (w/v) Paraformaldehyde (PFA). Slides were washed 3X with PBT (0.2% Triton X-100) before proceeding with antigen retrieval step.
Antigen Retrieval for IHC/IF: Slides were submerged in 10mM sodium citrate buffer (Na3C6H5O7, pH 6.0) containing 0.01% Triton X-100 at 95-98°C. Slides were kept in buffer at 95-98°C for 30 mins and then allowed to cool down to room temperature (RT).
Blocking and Antibody incubation for horseradish peroxidase (HRP)/3,3′diaminobenzidine (DAB) detection system (IHC): Slides were processed according to mCherry) compared to expressed proteins that localized in the cytosol (unique GFP or mCherry without DAPI signal). Fluorescent images are quantified for CCM1 using 488nm wavelength while CCM3 and PAQR8 were quantified using 555nm wavelength channel.
Imaging and Quantification for IHC: Imaging was carried out using a Nikon EclipseTi microscope with a color camera and a 10X objective lens. Quantification was conducted automatically using Elements Analysis software. Threshold was defined and maintained throughout all images for each application to ensure no bias was applied to the data.
Thresholds were applied to exclude low and high outliers. The red/brown color from the HRP/DAB reactivity with CCM2/PAQR7 antibodies was quantified and averaged between the red and green channel quantification.

Suppl. Fig. 1. Up-regulation of CCM and mPR proteins in breast cancers. Relative expression of total CCM proteins in paired-tissue breast cancer samples A).
Immunohistochemistry (IHC) approaches utilizing Horse Radish Peroxidase (HRP)/ 3,3′-Diaminobenzidine (DAB) staining revealed an increase in the relative intensity of CCM2 staining between representative breast tumor tissues compared to normal breast tissues (left panel); The corresponding, representative quantification is presented (middle panel) with statistical significance from the entire collection of paired samples (n=11, right panel) B). Significant increased protein expressions of CCM1 and CCM3 were visualized (left panel) in breast tumor (T), compared to normal tissue (N) samples using immunofluorescence (IF) imaging (n=3). C). Significant increased expression of PAQR7 was observed with HRP/DAB staining in breast tumors (Breast Carcinoma) from a selected set of breast tissue-pairs (left panel) with representative quantification displayed (middle panel). Statistically significant increased expression of PAQR7 (mPRα) was found in tumor tissues from the entire collection of paired samples (right panel, n=10). For HRP/DAB staining (panels A and C), the Red/Brown color from HRP/DAB reactivity is quantified and averaged between the red and green channel quantification. In fluorescent staining experiments (panel B), CCM1 and CCM3 were quantified through ROI intensities using wavelength channels 488 and 647nm, respectively. Data for both microscopy approaches were normalized against its respective internal controls using the blue channel for cell nuclei (HRP/DAB) or 408nm wavelength for DAPI (fluorescent) and background staining. For each section pair, Region of Interest (ROI) intensities were automatically quantified (over 1000 times/per section). All data from entire collections (n>10) were normalized by normal tissue among each tissue pair. All imaging data were PAQR8 staining inside the nucleus at 72 hrs compared to 0 hrs. B-1. Temporal modulation of CCM1 protein in T47D cells. T47D cells were treated with mPR-specific PRG treatment for 0-72 hrs and IF approaches revealed significant decreased expression observed after 4 hours in the relative intensity of CCM1 staining in T47D cells treated with mPR-specific PRG actions. B-2. Temporal modulation of CCM3 protein in T47D cells. IF approaches revealed decreased expression observed after 24 hours in the relative intensity of CCM3 staining in T47D cells treated with mPR-specific PRG actions. B-3.

Temporal modulation of PAQR8 (P8) protein in T47D cells. IF approaches revealed
increased expression observed at 24hrs in the relative intensity of PAQR8 staining in T47D cells treated with mPR-specific PRG actions.

Suppl. Fig. 7. Differentially expressed gene (DEG) detection utilizing RNAseq. A-B.
Cluster software and Euclidean distance matrixes were used for the hierarchical clustering analysis of the expressed genes (RNA) and sample program at the same time to generate the displayed Heatmap of hierarchical clustering for the intersection of DEGs Suppl.